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General Primer Design Guidelines
-Note the 5’-3’ direction of the contig.
-Locate primers 100 to 200 bp from the feature.
-Pick primer from ≥ 2x high quality sequence coverage.
-GC Clamp
-Avoid runs of identical nucleotides (e.g.
ATCGACCCCCTAGAC).
-Similar Tm ( /-2℃) for primers in same reaction set.
-Unique to the template.
Designing Primers forSequencing Gaps
-Length: 18 to 21 nucleotides
-Tm 56-60℃
-4 2 Rule:
Tm = (#G #C) x 4 (#A #T) x 2
-Inside the clone
Designing PCR Primers forPhysical Ends
-Length: 24 to 28 nucleotides
-Tm 62-66℃.
-Pairs with same Tm.
-Unique to template.
-Tm and self-complementarity:
http://www.basic.northwestern.eduhttp://www.bbioo.com/biotools/oligocalc.html
Unique Primer Alignment
Automated Primer Design using Primer3
Web-based or downloadable command-line tool.
Developed to design primers for PCR.
Designs primer pairs only.
Must concatenate adjacent contig ends to design gap primers.
Calculates Tm, self-complementaity, etc.
Available at:http://fokker.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
Scaffolds of the Sample Bac
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